In many assay technologies, it is necessary to use multiple enzymes to achieve an optimal assay. The goal of such an assay is to measure the activity of one enzyme (the target enzyme) on a substrate exclusively. However, once the target enzyme has catalyzed the reaction of interest, an additional enzyme or enzymes may be used to generate a signal that is easily quantifiable, can be measured in small amounts, or increases or decreases linearly with the activity of the enzyme.
An ideal assay would allow all substrates and enzymes to be combined in a single reaction mixture. However, additional enzymes must only react with the substrate of interest, and at the specified position on the substrate of interest, to prevent unwanted side reactions and allow the assay to monitor the activity of the target enzyme exclusively.